The following discussion of the background of the invention is merely provided to aid the reader in understanding the invention and is not admitted to describe or constitute prior art to the present invention.
Natriuretic peptides are a group of naturally occurring substances that act in the body to oppose the activity of the renin-angiotensin system. There are three major natriuretic peptides: atrial natriuretic peptide (ANP), which is synthesized in the atria; brain-type natriuretic peptide (BNP), which is synthesized in the ventricles; and C-type natriuretic peptide (CNP), which is synthesized in the brain.
Mature human A-type natriuretic peptide (ANP) (also referred to as atrial natriuretic peptide) is a biologically active 28 amino acid peptide that is synthesized, stored, and released by atrial myocytes in response to atrial distension, angiotensin II stimulation, endothelin, and sympathetic stimulation (beta-adrenoceptor mediated). Mature ANP is generated by proteolytic cleavage of a 128 amino acid precursor molecule (pro-ANP), yielding the biologically active 28 amino acid peptide representing amino acids 99-126 of the pro-ANP molecule (ANP99-126). Linear peptide fragments from the N-terminal prohormone segment have also been reported to have biological activity.
Mature human B-type natriuretic peptide (BNP) (also called brain-type natriuretic peptide) is a 32 amino acid, 4 kDa biologically active peptide that is involved in the natriuresis system to regulate blood pressure and fluid balance (Bonow, R. O., Circulation 93:1946-1950, 1996). The mature BNP hormone is generated by proteolytic cleavage of a 108-amino acid precursor molecule, referred to herein as “pro-BNP.” Cleavage generates t a 76-amino acid N-terminal peptide (amino acids 1-76), referred to as “NT pro BNP,” and the 32-amino acid mature hormone, referred to as BNP or BNP 32 (amino acids 77-108). It has been suggested that each of these species—NT pro-BNP, BNP-32, and the pre-pro-BNP—can circulate in human plasma (Tateyama et al., Biochem. Biophys. Res. Commun. 185:760-7, 1992; Hunt et al., Biochem. Biophys. Res. Commun. 214:1175-83, 1995).
Mature human C-type natriuretic peptide (CNP) a 22-amino acid peptide that is the primary active natriuretic peptide in the human brain; CNP is also considered to be an endothelium-derived relaxant factor, which acts in the same way as nitric oxide (NO) (Davidson et al., Circulation 93:1155-9, 1996). CNP is structurally related to A-type natriuretic peptide (ANP) and B-type natriuretic peptide (BNP); however, while ANP and BNP are synthesized predominantly in the myocardium, CNP is synthesized in the vascular endothelium as a precursor (pro-CNP) (Prickett et al., Biochem. Biophys. Res. Commun. 286:513-7, 2001). CNP is thought to possess vasodilator effects on both arteries and veins and has been reported to act mainly on the vein by increasing the intracellular cGMP concentration in vascular smooth muscle cells.
ANP and BNP are released in response to atrial and ventricular stretch, respectively, and will cause vasorelaxation, inhibition of aldosterone secretion in the adrenal cortex, and inhibition of renin secretion in the kidney. Both ANP and BNP will cause natriuresis and a reduction in intravascular volume, effects amplified by the antagonism of antidiuretic hormone (ADH). The physiologic effects of CNP differ from those of ANP and BNP; CNP has a hypotensive effect, but no significant diuretic or natriuretic actions. Increased blood levels of natriuretic peptides have been found in certain disease states, suggesting a role in the pathophysiology of those diseases, including stroke, congestive heart failure (CHF), cardiac ischemia, systemic hypertension, and acute myocardial infarction. See, e.g., WO 02/089657; WO 02/083913; and WO 03/016910, each of which is hereby incorporated in its entirety, including all tables, figures, and claims. Numerous non-human homologs of the natriuretic peptides are known to those of skill in the art.
The natriuretic peptides, alone, collectively, and/or together with additional proteins, can serve as disease markers and indicators of prognosis in various cardiovascular conditions. For example, BNP, which is synthesized in the cardiac ventricles and correlates with left ventricular pressure, amount of dyspnea, and the state of neurohormonal modulation, makes this peptide the first potential marker for heart failure. Measurement of plasma BNP concentration is evolving as a very efficient and cost effective mass screening technique for identifying patients with various cardiac abnormalities regardless of etiology and degree of LV systolic dysfunction that can potentially develop into obvious heart failure and carry a high risk of a cardiovascular event. Finding a simple blood test that would aid in the diagnosis and management of patients with CHF clearly would have a favorable impact on the staggering costs associated with the disease.
Removal of the natriuretic peptides from the circulation is affected mainly by binding to clearance receptors and enzymatic degradation in the circulation. See, e.g., Cho et al., Heart Dis. 1: 305-28, 1999; Smith et al., J. Endocrinol. 167: 239-46, 2000. Additionally, human pro-BNP is reported to be processed in serum such that circulating pre-pro-BNP is unlikely to be the intact 108 amino acid form. Hunt et al., Peptides 18: 1475-81, 1997. Degradation of the natriuretic peptides is believed mediated by neutral endopeptidase. For example, Norman et al. (Biochem. Biophys. Res. Commun. 28: 175: 22-30, 1991) report that neutral endopeptidase can cleave human BNP between residues 2 and 3, between residues 4 and 5, and between residues 17 and 18. Similarly, Lindberg and Andersson (Regul. Pept. 47: 53-63, 1993) report that human ANP is cleaved between residues 3 and 4 and residues 14 and 15. The biological activity of this hydrolyzed product was about 500-fold less than intact ANP. Additionally, Knecht et al. (Life Sci. 71: 2701-12, 2002) report that renal neutral endopeptidase is upregulated in heart failure, a condition where natriuretic peptide levels are increased. For this reason, neutral endopeptidase has been targeted for inhibition in treatment of cardiovascular disease. See, e.g., Corti et al., Circulation 104: 1856-62, 2001.
Confusion over the stability of the natriuretic peptides, particularly in blood-derived samples (e.g., serum, plasma, whole blood) has been reported. ANP is reported to be a better substrate for neutral endopeptidase than is BNP. Similarly, Shimizu et al. (Clin. Chem. Acta 305: 181-6, 2001), Gobinet-Georges et al. (Clin. Chem. Lab. Med. 38: 519-23, 2000) and Murdoch et al. (Heart 78: 594-7, 1997) report that BNP is stable in certain blood-derived samples or when blood is collected under certain conditions. A more recent report by Shimizu et al. (Clin. Chem. Acta 316: 129-35, 2002) indicates that 94% of BNP in whole blood was a digested form in which 2 amino terminal residues had been removed; and that BNP in plasma was degraded to a number of unidentified forms.